Pharmacological Evaluation of the Bronchorelaxant Effect of Waltheria Indica L

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Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2021, Article ID 5535727, 8 pages https://doi.org/10.1155/2021/5535727

Research Article Pharmacological Evaluation of the Bronchorelaxant Effect of Waltheria indica L. (Malvaceae) Extracts on Rat Trachea

Rainatou Boly ,1 Zakaline Yabre,2 Mathieu Nitiema ,1 Boubacar Yaro,1 Jules Yoda,1 Lazare Belemnaba ,1 Sylvain Ilboudo,1 Noe¨la Hoho Estelle Youl,2 Innocent Pierre Guissou,3 and Sylvin Ouedraogo1

1Institute of Research in Health Science (IRSS), 03 PO 7047, Ouagadougou 03, Burkina Faso 2Laboratory of Drug Development, Doctoral School of Sciences and Health, University Joseph KI-ZERBO, 03 BP 7021, Ouagadougou 03, Burkina Faso 3University of Saint 0omas D’Aquin, 06 PO 10212, Ouagadougou 06, Burkina Faso

Correspondence should be addressed to Rainatou Boly; [email protected]

Received 21 January 2021; Revised 10 April 2021; Accepted 15 April 2021; Published 27 April 2021

Academic Editor: Vincenzo De Feo

Copyright © 2021 Rainatou Boly et al. 'is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Waltheria indica L. (Malvaceae) is a plant used in Burkina Faso for the treatment of various ailments including asthma. 'e aim of the study was to evaluate the pharmacological relaxant effect of the leafy stem extracts of Waltheria indica and thereby verify claim of use in treating asthma. Aqueous decoction and hydroalcoholic extracts obtained from the powdered leafy stems were screened for the presence of some phytoconstituents. 'e in vitro relaxant effect of the two extracts was evaluated on acetylcholine- (ACh 10−5 M) and potassium chloride- (KCl 6 ×10−2 M) induced contractions on rat-isolated tracheal preparations. To examine whether the potassium (K+) channels are involved in the relaxant effect, glibenclamide, an ATP-sensitive potassium channel inhibitor, was used. Moreover, to assess the safety of the extracts, acute oral toxicity was carried out on mice. 'e phytochemical screening revealed the presence of alkaloids, flavonoids, saponins, steroids, triterpenoids, tannins, and coumarins in the hydroalcoholic extract. Tannins, steroids, triterpenoids, and coumarins were not detected in the aqueous decoction. With respective EC50 values of 1.517 ± 0.002 mg/mL and 1.433 ± 0.001 mg/mL on ACh-and KCl-provoked contractions, the hydroalcoholic extract was found more potent in relaxing the isolated rat tracheal preparations compared to the aqueous decoction. In the presence of glibenclamide, the relaxant effect of the hydroalcoholic extract (EC50 � 0.191 ± 0.002 mg/mL) increased and was higher than that of the aqueous decoction. At dose of 5000 mg/kg of body weight, the extracts did not produce deaths or any significant changes in the general behavior of mice. 'e results suggest that different mechanisms including modulation of calcium and potassium channels, particularly the ATP-sensitive K+ channels, could be involved in the relaxation effect. 'ese findings could justify the traditional use of W. indica in the management of asthma.

1. Introduction asthma affects more than 339 million persons, both children and adults, across the world regardless the level of devel- Asthma is a heterogenous disease characterized by multiple opment [2]. 'e prevalence of asthma varies from 1 to 18% factors involving genetic predisposition and environmental worldwide and the disease mostly affects poor children and factors [1–3]. It is one of the most common chronic diseases people among the general population [5, 9]. In fact, about that present different features including bronchocon- 250 000 deaths from asthma occur mostly in low- and striction, airway hyperreactivity, mucus secretion, and middle-income countries [2, 10]. Few studies have been chronic inflammation [4–6]. Asthma symptoms include dedicated to the study of asthma in Africa as reported by episodes of wheezing, coughing, and breath shortness [7–9]. Adeloye et al. [10]. In their report, the number of asthma 'e World Health Organization (WHO) estimates that cases is estimated at 119.3 million in 2010 [10]. Asthma 2 Evidence-Based Complementary and Alternative Medicine represents a public health problem that is underdiagnosed W. indica has many other names. Hence, it is called “Nal- and undertreated, with the consequence of increasing the labenda” in Telugu, “kafafi” in Fulani, “hankubath” in disease burden, mainly in least developed countries Hausa, “korikodi” in Yoruba, “matum kevel” in Wolof, “yar- [2, 9, 10]. yamde” in Moore, “Mokhutesela” in South Africa, “guinar¨ ” According to the Global Initiative of Asthma (GINA), in Mexico, “uhaloa” in Hawaii, and “malvavisco” in Spanish the treatment of asthma aims to control symptoms in order [22]. Waltheria indica is a short-lived shrub reaching 2–7 m to minimize risk of exacerbations [1]. Numerous com- height and a stem diameter of 2 cm. 'e plant is widely pounds have been developed for the management of asthma. present in subtropical and tropical regions and can grow on 'ese compounds act to (i) reverse or prevent bronchial many areas including roadside weed, old pastures, inun- smooth muscle constriction and (ii) reverse or prevent dated savannas, or riverbanks [23, 24]. Different parts of this airway inflammation [11]. 'e current approach to the plant are customarily used to treat diverse ailments in- treatment of asthma consists of using corticosteroids (oral cluding pain, cancer, inflammation, cough, sore throat, lung and inhaled) associated with bronchodilators (short- and infections, and asthma [23, 25–27]. Ethnobotanical data long-acting beta 2-agonists), leukotriene antagonists, and gathered from traditional healers confirmed the use of the long-acting anticholinergics [6, 7, 9, 12]. Nevertheless, de- aerial parts, roots, and whole plant of Waltheria indica to spite the fact that most current antiasthmatic drugs have treat asthma in Burkina Faso [16]. Preclinical studies in- proven to be effective, the need to develop or search for new dicated that Waltheria indica and its active compounds ones is necessary. Indeed, the current asthma drugs are exhibited analgesic, anti-inflammatory, cancer chemo- associated with the occurrence of several side effects preventive effect, antioxidant, antidiabetic, antiviral, anti- [6, 13, 14]. For instance, the long use of inhaled cortico- bacterial, bronchorelaxant, and other potential steroids may be accompanied by bronchitis, nasophar- pharmacological activities [23, 25, 28–31]. 'e pharmaco- yngitis, and respiratory tract infection while the common logical effect of W. indica is based on the presence of nu- adverse effects of anticholinergic drugs (beta-2-agonists) are merous bioactive compounds such as tannins, flavonoids, urinary tract infection, dyspnea, headache, cough, and terpenes, alkaloids, carbohydrates, sterols, cardiac glyco- nausea [14]. Furthermore, these drugs are sometimes not sides, and anthraquinones [22]. accessible to many patients, mainly in poor countries, due to Few reports have been dedicated to the study of the use their high cost. 'e annual management of asthma in terms of Waltheria indica L. for the management or treatment of of both direct and indirect costs in Europe and United States asthma. 'erefore, the present study was undertaken to of America is approximately estimated at €18 billion and investigate the pharmacological relaxant effect of the leafy US$13 billion, respectively [3]. According to a recent report stem extracts of Waltheria indica on rat trachea. Further- on the economic burden of asthma, annual direct costs more, this study aims to provide an attempt to explain the varied from US$150 to US$3,000 per patient [15]. In 2005, mechanism of the bronchorelaxant action. the annual treatment of a moderate asthma costed approximately US$192 in Burkina Faso [16]. Due to the several 2. Materials and Methods drawbacks associated with the conventional treatment of asthma, the development of novel therapeutic approaches 2.1. Chemicals and Reagents. All chemicals and reagents effective at low cost and with low side effects will alleviate used to carry out the experiments were of analytical grade. asthma patients. Acetylcholine was purchased from Sigma-Aldrich (St. Louis, 'e use of phytomedicine or traditional herbal medi- MO). Potassium chloride (KCl) was supplied by Labosi cines is one of these approaches. Herbal medicines are (France). Tablets of 5 mg glibenclamide (Tongmei, Togo) widely used worldwide due to their important health benefits were purchased at a local pharmacy in Ouagadougou, capital combined with reduced adverse effects and toxicity city of Burkina Faso. Dichloromethane, n-hexane, and [3, 17–19]. Indeed, according to the WHO, at least 80% of butanol were obtained from Carlo-Erba (France). Ethanol African populations rely on traditional herbal medicine for and ethyl acetate were procured from Prolabo (France). their primary healthcare due to its accessibility and af- Silica gel TLC plates F 254 grade were from Macherey-Nagel fordability [17, 19, 20]. (Germany). Several authors reported the large and successful use of herbal medicine among both adults and children suffering from asthma [3, 7, 18, 21]. However, despite these significant 2.2. Equipment. 'e apparatus used to achieve the bron- findings, it is noteworthy that the pharmacological valida- chorelaxant effect of extracts from leafy stems of Waltheria tion of a number of these plant-derived medicines still needs indica includes the following: an isolated organ study device to be demonstrated. equipped with four organ bath chambers (of 20 mL each), In an effort to provide significant pharmacological in- isometric transducers (Emka Technologies, France) for formation about antiasthmatic plants, this study focused on measuring isometric force, an amplifier (Emka Technolo- Waltheria indica L. (Malvaceae) (syn. Waltheria americana). gies, France) for recording changes in isometric force, and a 'e common and vernacular names of W. indica have been computer for data acquisition. Other instruments used in- intensively reported [22]. 'e plant is commonly named clude water bath (Julabo, GmbH, Germany), analytical sleepy morning, monkey bush, marsh-mallow, boter bush, balance (Sartorius, Germany), oven (Memmert GmbH, or velvet leaf. Depending upon the geographic region, Germany), and rotary evaporator (Buchi, Switzerland). Evidence-Based Complementary and Alternative Medicine 3

2.3. Plant Material. Leafy stems of W. indica were collected 423 [36]. Each animal was treated once with a single oral from the area of Gomboussougou located in the Centre- dose of 5000 mg/kg of extract by gavage using a feeding tube. South region of Burkina Faso (11 25′ 15, 12 N 0 45′ 32, 4 W). Prior to the administration of extracts, mice were weighted, 'e plant was taxonomically authenticated and a voucher marked, and fasted for 3–4 h, but with access to water. specimen bearing the number ID 16876 was preserved at the Briefly, four groups of three mice each were constituted. 'e herbarium of the Laboratory of Plant Biology and Ecology, 1st and 2nd groups received orally a single dose of 5000 mg/kg University Joseph Ki-Zerbo, Burkina Faso. of body weight of aqueous decoction (AD) and hydroalcoholic extract (HE), respectively. 'e 3rd and 4th were controls groups for AD and HE, respectively; the mice of 2.4. Preparation of the Plant Extracts. 'e leafy stems of these groups received distilled water and a 5% aqueous Waltheria indica were shade-dried under room temperature ethanol solution. Two hours after the administration of the and powdered with a grinder. Two extracts were prepared extracts, mice were given ad libitum access to food and from the powdered leafy stems. water. 'ereafter, the experiment was repeated with male 'e aqueous decoction was obtained by bringing to boil mice (control and tested groups). 150 g of grounded leafy stems mixed with 1.5 L of distilled 'e health condition of the mice was observed each water for 1 h. 'en, the extract was filtered and freeze-dried. 30 min during the first two hours following the treatment To prepare the hydroalcoholic extract, 150 g of powdered and then, twice a day for 13 days. Mice were monitored to leafy stems was macerated in a water and ethanol mixture report any possible clinical signs of toxicity including (1.5 L) in proportion of 20 : 80 (v/v) at room temperature and mortality, change in general behavior, movements, and under mechanical stirring for 48 h. 'ereafter, the extract reflexes. was filtered, concentrated using a rotary evaporator, and lyophilized. 'e residual moisture’s content of the powder and the extraction’s yield of the two extracts were deter- 2.8. Bronchorelaxant Study mined in accordance with the literature [32, 33]. 'e two lyophilized extracts were then kept in a freezer 2.8.1. Preparation of Tracheal Rings. 'e bronchodilation until required for the biological assays. effect of the two extracts from Waltheria indica leafy stems was assayed on rat isolated tracheal preparations according 2.5. Preliminary Phytochemical Investigation. 'e powdered to a modified method of Ouedraogo et al. [37]. Rats were leafy stems of Waltheria indica as well as the decoction and starved 24 h prior to starting the experiment and anes- hydroalcoholic extracts were screened for the presence and/ thetized with ketamine (1 g/kg of body weight). 'e animals’ or absence of various secondary metabolites including fla- chest was opened and the trachea was excised and placed vonoids, tannins, alkaloids, saponins, coumarins, steroids, quickly in a Petri-dish containing a modified Krebs-Hen- and triterpenoids, using suitable standard qualitative seleit physiological solution (having composition (in mM) of methods [34]. 'e thin layer chromatography was carried NaCl 118; NaHCO3 24.1; KCl 4.7; KH2PO4 1.2; MgSO4 2.5; out on precoated plates using two solvent systems: (i) CaCl2 3.33; Glucose 3.33 and the pH was adjusted to 7.4). hexane/ethyl acetate/methanol (14/4/2, v/v/v) and (ii) ethyl 'e trachea was separated from surrounding tissue and cut acetate/methanol/water (20/2, 6/2, v/v/v). Specific spray into four to five rings of about 5 mm in length. Each ring was reagents highlighted the presence of the main mounted in a 20 mL organ bath chambers containing a phytoconstituents. modified Krebs-Henseleit physiological solution, continuously aerated, and maintained at the temperature of 37°C. Tracheal isolated rings were allowed to equilibrate under a 2.6. Experimental Animals. NMRI strains mice (20–30 g) and tension of 1 g for 1 h, a time during which bathing solution Wistar strains rats (300–400 g) of either sex were obtained from was replaced every 15 min. the animal facility of the department of Medicine, Traditional Pharmacopeia, and Pharmacy at the Institute of Research in Health Sciences (IRSS). 'e animals were maintained under 2.8.2. Effect of Acetylcholine (ACh) on the Isolated Rat standard laboratory conditions (temperature of 22 ± 3°C, 12/ Trachea. Before commencing the experiment, KCl at 80 mM 12 h light/dark cycle and relative humidity of 50–70%) with ad was added in the bath during the plateau phase to assess the libitum access to food and water. However, before the ex- contractile reactivity of tracheal rings. 'en, the tracheal periment, food was drawn back but the animals still had free rings were washed several times with fresh Krebs-Henseleit access to water. Experimental protocols were strictly performed physiological solution, left to recover, and returned to their in accordance with the eighth edition of the “Guide for the Care initial state. 'ese steps of precontraction with the KCl and Use of Laboratory Animals” with minimal number of followed by several washes were continuously repeated animal usage according to the 3Rs principle, i.e., Replacement, before the start-up of each experimental procedure. Reduction, and Refinement [35]. In order to determine the concentration of acetylcholine that induces maximal contraction of the tracheal ring, cu- 2.7. Acute Toxicity Study. To conduct the acute toxicity mulative concentrations of ACh (10−6 to 1.5 ×10−5 M) were assay, a limit test at 5000 mg/kg of extract was used in both added in the organ bath. A maximal contraction effect was male and female mice according to the OECD Guidelines obtained with a concentration of ACh equal to 10−5 M. 4 Evidence-Based Complementary and Alternative Medicine

2.8.3. Effect of Waltheria indica Leafy Stem Extracts on the decoction and hydroalcoholic extract elicited an interesting Isolated Rat Trachea. 'e effects of AD and HE were bronchorelaxant response with respective EC50 values of evaluated on trachea rings precontracted with either ACh 1.517 ± 0.002 mg/mL and 1.200 ± 0.002 mg/mL. 'e maxi- 10−5 M or KCl 6 ×10−2 M, by cumulative addition of the mal bronchorelaxant effect (Emax) was equal to extracts at the concentrations of 10, 30, 100, 300, 1000, 2000, 88.54 ± 12.44% and 88.86 ± 10.38%, respectively, for the and 3000 μg/mL. aqueous decoction and the hydroalcoholic extract. 'ere To study whether potassium channels are involved in the were no statistically significant differences between the two mechanism of the bronchorelaxant effect, glibenclamide, an extracts when tested on acetylcholine-induced contractions. ATP-sensitive potassium channel inhibitor [38], was used. In tracheal rat rings precontracted with KCl 6 ×10−2 M Briefly, glibenclamide at 10−5 M was added in the organ bath (Figure 1(b)), the aqueous decoction and the hydroalcoholic followed 15 min later with ACh 10−5 M. 'en, the test ex- extract produced a maximal bronchorelaxant effect of tracts were cumulatively added at the respective concen- 48.42 ± 9.71% and 89.53 ± 18.06%, respectively. 'e hydro- trations of 10, 30, 100, 300, 1000, 2000, and 3000 μg/mL. alcoholic extract significantly inhibited the KCl-induced contractions and was more potent with an EC50 value of 1.433 ± 0.001 mg/mL. 'e EC value of the aqueous de- 2.9. Data Presentation and Statistical Analysis. Signs (+) or 50 coction was slightly higher than 3 mg/mL (the maximum (-) were used to assess the presence or not of phytochemical concentration tested) and was equal to 3.159 ± 0.001 mg/mL. groups in the tested extracts. 'e percentage of relaxation and the EC50, i.e., the concentration required to induce 50% of the maximal bronchorelaxant effect, were expressed as 3.3.2. Effect of Waltheria indica Leafy Stem Extracts on � mean ± SD (standard deviation) of four experiments (n 4). Acetylcholine-Induced Contractions Response of Isolated Rat 'e EC50 values and the plotted curves of the bronchor- Trachea in the Presence of Glibenclamide. Glibenclamide elaxant effect were obtained with GraphPad Prism® Software was used to study whether potassium channels are involved (version 6.07). Data were compared by a two-way ANOVA in the mechanism of the bronchorelaxant effect of extracts. with the Bonferroni correction for multiple comparisons. 'e tracheal rings were pretreated with glibenclamide at p −5 −5 Differences were considered significant with a value <0.05. 10 M before addition of ACh 10 M in the organ bath 15 min later and then by test extracts. Figure 2 presents the 3. Results relaxant effect of Waltheria indica leafy stem extracts on acetylcholine-precontracted rat tracheal rings in presence of 3.1. Preliminary Phytochemical Investigation. 'e yield of glibenclamide. 'e relaxant effect of the aqueous decoction the extracts, the residual moisture content, and the results of was significantly reduced in presence of glibenclamide. the preliminary phytochemical search of Waltheria indica Indeed, in absence of glibenclamide, the pharmacological leafy stem extracts are summarized in Table 1. parameters, namely, the Emax and EC50, vary from 88.54 ± 12.44% to 21.09 ± 8.12% for the Emax and from 3.2. Acute Toxicity Study. 'e acute toxicity was assessed in 1.517 ± 0.002 mg/mL to 4.722 ± 0.001 mg/mL for the EC50 mice according to the OECD Guidelines 423. 'e results values. Pretreatment of the tracheal rings with glibenclamide showed no mortality at the limit dose of 5000 mg/kg of body in presence of the hydroalcoholic extract significantly weight. Apart from a slight drowsiness observed during the (p < 0.0001) increased the Emax (from 88.86 ± 10.38% to first 30 min following the administration of the plant ex- 109.88 ± 5.47%) and decreased the EC50 value from tracts, there were no significant changes in the general 1.200 ± 0.002 mg/mL to 0.191 ± 0.002 mg/mL. behavior of the extract-treated mice. 'e administration of the hydroalcoholic extract (HE) decreased mice body weight gain although this was minor. In contrast, the aqueous 4. Discussion decoction (AD) caused a slight increase of mice body weight Waltheria indica L. (Malvaceae) has been claimed to treat at the end of the experiment period (14 days). various ailments including asthma. 'is study was undertaken to provide scientific evidence regarding the use of the 3.3. Bronchorelaxant Study leafy stems in the management of asthma. 'e yield of the aqueous decoction extraction was higher than that of the 3.3.1. Effect of Waltheria indica Leafy Stem Extracts on hydroalcoholic extract. 'is could be due to the effect of heat Acetylcholine- and Potassium chloride- (KCl-) Induced which contributed to extract more compounds compared to Contractions Response of Isolated Rat Trachea. Figure 1 the compounds in the hydroalcoholic extract that were presents the relaxant effect of Waltheria indica leafy stem extracted by maceration technique. As recommended, the extracts on acetylcholine- and KCl-precontracted rat tra- moisture content was less than 10% suggesting that the cheal rings (Figures 1(a) and 1(b), respectively). 'e aqueous powdered leafy stems of Waltheria indica are well preserved, decoction and hydroalcoholic extract from the leafy stems of without risk of contamination and/or alteration of the W. indica dose dependently inhibited the contraction re- phytochemicals [39]. sponse in tracheal rings contracted by acetylcholine or 'e powdered leafy stems and the hydroalcoholic extract potassium chloride (KCl). In tracheal rings precontracted contain alkaloids, flavonoids, tannins, triterpenoids, ste- with acetylcholine, 10−5 M (Figure 1(a)), the aqueous roids, coumarins, and saponins. 'ese results corroborate Evidence-Based Complementary and Alternative Medicine 5

Table 1: Preliminary phytochemical study results of Waltheria indica leafy stem extracts. Plant part/extract Phytochemical compound Powdered leafy stems Aqueous decoction Hydroalcoholic extract Flavonoids + + + Saponins + + + Alkaloids + + + Tannins + − + Steroids + − + Triterpenoids + − + Coumarins + − + Residual moisture content (%) 5.83 ± 0.02 n.d. n.d. Extract yield (%) 22.21 ± 0.08 14.54 ± 0.02 Abbreviations: (+) present; (−) absent; n.d.: not determined.

log C (µg/mL) log C (µg/mL) 1.0 1.5 2.0 2.5 3.0 3.5 4.0 1.0 1.5 2.0 2.5 3.0 3.5 4.0 –25 –25

0 0

25 25 ∗∗∗ 50 50 ∗∗ 75 75 Relaxation (%) Relaxation (%) 100 100

125 125

AD AD HE HE (a) (b)

Figure 1: Relaxant effect of the aqueous decoction (AD) and hydroalcoholic extract of Waltheria indica leafy stems on acetylcholine (a) and KCl (b) contracted rat tracheal rings. Data and error bars represent the mean ± SD of four independent experiments. Statistical comparisons between the two extracts were assessed using a two-way ANOVA with Bonferroni multiple comparison test. ∗∗∗p < 0.001 and ∗∗p < 0.001 (n � 4) indicate the statistical differences. previous studies reviewed by Nirmala and Sridevi [22] and indica exhibited significant concentration-dependent re- Zongo et al. [23]. However, we noticed the absence of laxant effects. 'e hydroalcoholic extract was more potent at tannins, coumarins, steroids, and triterpenoids in the inhibiting the contractions induced by ACh and KCl than aqueous decoction. 'e absence or presence of a particular the aqueous decoction. 'ese results showed different phytoconstituent in an extract depends on its solubility in mechanisms of bronchorelaxant effect. As reported else- the extraction solvent (water/ethanol) or on the extraction where [43], contraction of the smooth muscle is mainly method (maceration/decoction) [40]. Consequently, the linked to the increase of the concentration of intracellular 2+ 2+ absence of some secondary metabolites in the aqueous calcium ([Ca ]i). [Ca ]i can originate from extracellular decoction probably means that these substances have been space and intracellular store, especially from the sarco- destroyed during the extraction process. plasmic reticulum. KCl induced contraction by increasing 2+ Interestingly, the results of the acute toxicity showed intracellular calcium ([Ca ]i) through voltage-dependent that, up to 5000 mg/kg of body weight, no mortality or any Ca2+ channels while acetylcholine activates a G-protein- signs of toxicity were observed during the observation pe- coupled and produces contraction by increasing the Ca2+ riod. 'is result suggests that the extracts were less toxic and sensitivity and Ca2+ entry via the voltage-dependent Ca2+ therefore the lethal dose (LD50) was considered to exceed channels and receptor-operated calcium channels [44]. 'e 5000 mg/kg. 'e weight loss, though minor observed with receptor-operated calcium channels are coupled to mus- the administration of the hydroalcoholic extract at the end of carinic receptors and their activation caused the release of the test period, may be attributed to the presence of tannins Ca2+ from the sarcoplasmic reticulum [43, 44]. 'e ability of and saponins, phytoconstituents known to modulate body hydroalcoholic extract to inhibit the KCl- and ACh-induced weight [41, 42]. contractions suggests that it may decrease Ca2+ influx and/or In rat tracheal rings precontracted with KCl and ace- block the receptor-operated calcium channels which in turn tylcholine, the extracts from the leafy stems of Waltheria affect the release of Ca2+ from the sarcoplasmic reticulum. 6 Evidence-Based Complementary and Alternative Medicine

log C (µg/mL) 1.0 1.5 2.0 2.5 3.0 3.5 4.0 - 25

0 ∗∗∗∗

25 ∗∗∗∗ ∗∗

Relaxation (%) ∗∗∗∗ 75

100

∗∗∗∗ 125 ∗∗∗∗

AD + GLIB HE + GLIB Figure 2: Relaxant eﬀect of the aqueous decoction (AD) and hydroalcoholic extract of Waltheria indica leafy stems on acetylcholine- induced contractions in rat tracheal rings in the presence of glibenclamide (GLIB). Data and error bars represent the mean ± SD of four independent experiments. Statistical comparisons between the two extracts were assessed using a two-way ANOVA with Bonferroni multiple comparison test. ∗∗∗∗p < 0.0001 and ∗∗p < 0.01 (n � 4) indicate the statistical diﬀerences.

However, the bronchorelaxant effect of the aqueous de- previously proven and they exert their antiasthmatic effects coction may be explained by an effect on the receptor-op- through bronchodilatation, inhibition of Ca2+ channels, and erated calcium channels (ACh-induced contraction) instead blocking of muscarinic receptors [3, 46, 47]. Zongo et al. [29] of an effect on the voltage-dependent Ca2+ channels (KCl- have isolated epicatechin from the hydroalcoholic extract of induced contraction). Waltheria indica roots. 'is flavonoid was found to be 'e extracts were tested on acetylcholine contracted rat potent on smooth muscle relaxation and at inhibiting 5-Lox trachea preparation in presence of glibenclamide to study enzyme activity, two important targets for asthma therapy whether potassium channels are involved in the mechanism [29]. 'e relaxant effect of aqueous decoction, though less of the bronchorelaxant effect. 'ere are five types of po- potent compared to that of hydroalcoholic extract, may be tassium (K+) ion channels that play important roles in the due to the presence of flavonoids, alkaloids, and saponins regulation of vascular smooth muscle tone. Indeed, their [3]. activation produced membrane hyperpolarization leading to vasodilatation, whereas their inhibition caused membrane 5. Conclusion depolarization and thereby vasoconstriction [45]. In the presence of glibenclamide, an ATP-sensitive potassium Following the antiasthmatic claim of Waltheria indica, the channel inhibitor (K+-ATP channel), the relaxant effect of present study was undertaken to study the bronchorelaxant the hydroalcoholic extract was higher than in the absence of effect of the leafy stems of Waltheria indica (Malvaceae). 'e this inhibitor. 'is result implies that the hydroalcoholic hydroalcoholic extract stands to be more potent compara- extract may induce relaxation through different pathways tively to the aqueous decoction at inhibiting the contractions 2+ including the decreasing of [Ca ]i, the blockade of receptor- induced by both acetylcholine and potassium chloride. 'e operated calcium channels, and the opening of K+-ATP significant effect of the hydroalcoholic extract may be due to channels despite the presence of glibenclamide. However, the presence of various phytoconstituents which work the relaxant effect of the aqueous decoction was significantly synergistically through different mechanisms of action to reduced in the presence of glibenclamide, suggesting the exert the relaxant effect. Furthermore, we hypothesized that involvement of the K+-ATP channels in its relaxation the relaxant activity of the aqueous decoction may involve mechanism. the potassium ion channels, particularly the ATP-sensitive + 'e major phytoconstituents of the hydroalcoholic ex- K channels (KATP). However, further studies are needed to tract include alkaloids, flavonoids, tannins, triterpenoids, explore other mechanisms of the bronchorelaxant effect and steroids, coumarins, and saponins. 'e antiasthmatic carry out the chemical fingerprinting of the leafy stems of properties of most of these phytochemicals have been Waltheria indica. Evidence-Based Complementary and Alternative Medicine 7

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